Most functional deficits after spinal cord injury are caused by the disruption of nerve fibers that project longitudinally and interconnect the brain and spinal cord. In principle, there are two possible strategies for re-building functional circuits to repair this loss of communication: nerve fibers that were not damaged can be stimulated to sprout collateral axons and build compensatory connections, and injured axons can be stimulated to grow across the lesion to reconnect with their original targets. Major progress has been made recently in promoting sprouting and regeneration of the corticospinal tract (CST), a major pathway for controlling movement. It is not known;however, if these sprouted and regenerated CST axons can re- establish functional synaptic connections. This proposal addresses that second step, determining if sprouting or regenerating CST axons can make functional synaptic contacts with their normal target neurons in the spinal cord. Recent studies have shown that genetic deletion of PTEN in CST neurons results in robust contralateral sprouting of their axons in the spinal cord following ablation of the contralateral CST and also promotes unprecedented regeneration of CST axons across spinal cord lesions. It remains unknown, however, if sprouted or regenerated axons can make functional synaptic connections. A major target of CST axons in mice are Clarke's column neurons, located in the C11 - L2 segments of the spinal cord, in close proximity to the CST. We propose to utilize in vivo electrophysiological approaches to assess the ability of re-growing CST axons form functional synapses with Clarke's column neurons. In the first aim, we will induce the axons in one CST to sprout into the contralateral spinal cord by interrupting the other CST via a unilateral pyramidotomy in PTEN-deleted mice. We will test if sprouted CST axons establish functional synaptic connections by selectively stimulating the CST while recording intracellularly from Clarke's column neurons. We can thus test if axonal sprouts can form functional synapses with appropriate synaptic targets in the spinal cord. In the second aim, the CST will be lesioned bilaterally by a complete spinal cord crush at T10. This surgical procedure interrupts all axons that project through the crush region. After allowing CST axons to regenerate through the lesion in PTEN-deleted mice, we will record intracellularly from Clarke's column neurons just caudal to the crush while stimulating the CST at cervical levels above the crush. These experiments will test if CST axons regenerating through the lesion are able to form functional synaptic connections below the lesion. Taken together, these experiments will allow us to assess an important functional aspect of sprouting and regenerating CST axons, namely their ability to form functional synaptic connections. These results should provide direct insights into designing therapeutic strategies for re-establishing corticospinal connections and promoting functional recovery after spinal cord injuries. PUBLIC HEALTH RELEVANCE: Spinal cord injuries cause a major loss of function because nerve fibers that connect the brain and spinal cord are disrupted at the site of injury. Major progress has been made recently in promoting re-growth of the corticospinal tract (CST), a major pathway for controlling movement, across the injured site, but it is unknown if CST nerve fibers can re-establish functional connections with appropriate nerve cells below the injury. The proposed experiments will use electrical recordings from mouse spinal cords to determine if re- growing CST nerve fibers can form functional connections with these nerve cells, thus providing insights into designing therapeutic strategies for promoting functional recovery after spinal cord injuries.